Allergenic response induced by Pterobothrium crassicolle (Cestoda: Trypanorhyncha) extracts in murine model

Abstract The aim of this study was to determine the allergenic activity of components present in crude extracts of Pterobothrium crassicolle plerocerci (CPE) and blastocysts (CBE) obtained from Micropogonias furnieri in a murine model. Two groups of seven animals each received 50 µg of CPE or CBE on days 1, 35 and 120. Serum samples were tested by ELISA and Immunoblotting. Specific IgG and IgE levels were detected by ELISA, showing specific humoral responses for the primary immunization for both immunoglobulins and continuously growing titers for IgE. Positive Passive Cutaneous Anaphylaxis tests in rats sensitized with anti-CBE sera and tested by CBE, showed biologically, the allergenic activity of the extracts. The CPE and CBE showed some different recognition regions but both experimental groups recognized all regions of the extracts when tested for cross reactions, showing that CPE and CBE could share antigenic recognition sites.

Due to the increasing worldwide consumption of raw, undercooked or poorly processed fish, human accidental infections with fish parasites and some allergic related reactions have represented a serious public health hazard, with increasing medical concern in several countries (Chai et al., 2005;Audicana & Kennedy, 2008;Dorny et al., 2009;Broglia & Kapel, 2011).Human parasitism by trypanorhynch cestodes is extremely rare (Kikuchi et al., 1981;Fripp & Mason, 1983), however Pelayo et al. (2009) showed the seroprevalence of an immune response against the trypanorhynch Gymnorhynchus gigas in a Spanish population.According to Deardorff et al. (1984), the metacestode toxins are gradually released to the fish tissues, mostly flesh, which could represent a hazard for human health, and experimental studies have highlighted the risk of allergic reactions by trypanorhynchs (Rodero & Cuéllar, 1999;Vázquez-López et al., 2001, 2002;Gòmez-Morales et al., 2008;Mattos et al., 2015).
Considering the lack of data about the allergenic potential of Pterobothriidae trypanorhynchs, the aim of the present study was to determine if crude extracts of Pterobothrium crassicolle (Diesing, 1850) plerocercoids and blastocysts have antigenic compounds able to induce specific allergic responses in experimental murine model.

Material and Methods
A total of 107 specimens of M. furnieri (24.0-65.0cm) were obtained from fish markets and fishermen in the municipalities of Niterói and Cabo Frio, Rio de Janeiro State, Brazil, between March/2009 and March/2012.They were collected and transported on ice in isothermic bags for examination at the Laboratório de Inspeção e Tecnologia de Pescado, Faculdade de Veterinária (Fish Inspection and Technology Laboratory, Faculty of Veterinary), Universidade Federal Fluminense (UFF).The fish specimens were identified according to Menezes & Figueiredo (1980) and submitted to necropsy at the laboratory.Parasite recovery was carried out according to the methodology proposed by Eiras et al. (2006).The taxonomic identification of trypanorhynch cestodes was based on Campbell & Beveridge (1996) and identified as P. crassicolle metacestode.The plerocerci of P. crassicolle and its blastocysts were manually collected from the fish with the aid of scissors and forceps.
The metacestodes were transported on ice inside isothermic bags to the Laboratório de Imunobiologia das Doenças Infecciosas e Granulomatosas, Departamento de Imunologia, Instituto de Biologia (Department of Immunobiology, Institute of Biology), UFF, where immunological analyses were carried out.The crude plerocerci extract (CPE) and the crude blastocysts extract (CBE) were obtained after separation of the metacestode parts in different containers, followed by extensive washing using sterile 0.1M phosphate-buffered saline (PBS), pH 7.3, supplemented with 5% penicillin and 5% streptomycin.The metacestode parts were homogenized singly in a Potter-Elvehjem homogenizer (Thomas Scientific, PA, USA) after a final wash with non-supplemented, sterile PBS.The homogenate was then submitted to six 30-s cycles using the Tissue Ruptor (Qiagen Instruments AG, Zurich, Switzerland), the suspension obtained centrifuged at 60.000 g at 4ºC for 30 minutes and the supernatant filtered through a 0.22 µm MillexGV Millipore filter (Millipore, France).
The same protocol was used to prepare the crude fish protein extract (CFE) of M. furnieri, which was used as the control antigen for the serological assays.The protein contents of the CPE, CBE and CFE were estimated according to Lowry et al. (1951).
To determine the molecular weight range of the CPE, 0.03mg of the extract was submitted to SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) using a 12%, 100 x 100mm gel (Vertical System, Bio-Rad, Hercules, California, USA) for 2h at 140V (Laemmli, 1970).
Ten-week-old female BALB/c mice were maintained in separate cages according to their experimental group (two experimental groups [n=7] and control group [n=5]), receiving distilled water and food (Nuvilab CR-1, Nuvital Nutrientes S/A, Brazil) ad libitum.All animals were injected with xylazine (200 μg/kg g/kg body weight) intramuscularly associated with ketamine (10 mg/kg body weight) before invasive procedures.Euthanasia was applied using an overdose of anesthetic drugs.The study was approved by the Animal Research Ethics Committee of the UFF Centre for Laboratory Animals (038∕2009).
Each experimental group was immunized intraperitonally (i.p.) on days 1, 35 and 120, with a suspension containing 50 µg of CPE or CBE and 2.0 mg of commercial aluminum hydroxide solution, Al(OH) 3, in a final volume corresponding to 200 μl of suspension.At the same times the control group was injected with a suspension containing only sterile saline and aluminum hydroxide.
Six female Lou-M adult rats each weighing 150g were reared in the animal house of the UFF and tested using the Passive Cutaneous Anaphylaxis (PCA) assay.This technique, as described by Braga & Mota (1976), uses a 72 h sensitization period for the IgE antibody.Briefly, a shaved dorsal area was injected intradermally with 30 μL of mice sera from the CPE, CBE or control groups (days 56, 120, 127 and 135) diluted 1:40.After the sensitization period, PCA reactions attributable to the IgE class were elucidated by the rats by the intravenous administration of 500 μg of CPE, CBE or CFE in 0.5 mL saline mixed with 1% Evans blue dye.Saline (0.5 mL) was used as the negative control.Thirty minutes later, the rats were euthanized by an overdose of anesthetic drugs.The dorsal skin was removed and inverted to observe and measure any pigmented area and the reactions considered positive for spots larger than 5 mm in diameter.
The recognition of immunogenic proteins by Immunoblotting (Western Blot) was used to determine the reactivity profile of specific IgG and IgE.For the western blot, 0.3 mg of CPE and CBE were submitted to the same SDS-PAGE conditions, followed by transfer of the protein bands from the separating gel to the nitrocellulose membrane using a Semi-dry blotter (Bio-Rad, CA, USA).Subsequently, the membranes were blocked overnight with 5% fatfree milk (Nestle) in PBS solution, washed with 0.05% PBS-Tween, dried at room temperature (RT) and cut into strips.Two strips were incubated overnight at RT with each serum sample diluted 1:100 v/v in blocking buffer, Immunogenic activity of Pterobothrium crassicolle with constant rocking.After washing four times with TBS-(Tris-buffered saline) Tween, one membrane strip of each serum was incubated with peroxidase-labelled goat anti-mouse IgG (Bio-Rad) for 2h and the other exposed to rat anti-mouse IgE (Invitrogen) for 3h, followed by HRP-goat anti-rat IgG (H + L, Bio-Rad, CA, USA) for 2h at RT with constant rocking.After at final wash, the peroxidase substrate (3.3'-diaminobenzidine,Sigma-Aldrich, USA) was added to develop the Ag/IgG or Ag/IgE interaction.All antibodies were used according to the manufacturer's recommendations.
The Shapiro-Wilk test was used to assess normality.Data were evaluated using the General Linear Model, with repeated measures ANOVA and Bonferroni post-hoc.The software used was SPSS (IBM, version 24).In the statistical analysis of experimental data, the values were considered significant at p< 0.05.

Results
After the primary immunization, specific IgG and IgE were detected in the serum samples of the experimental groups as from day 14, with statistically significant increasing levels (p<0.001 for all, except for IgE of the CBE group, which was p<0.01 at day 14) when compared with the control group sera.The highest IgG level was observed for the samples collected on day 42 from animals immunized with 50µg of CBE.The titers of specific IgE increased continuously for both the CPE and CBE groups during the experimental period (Figure 1).Cross-reactions between the immunized groups (CPE and CBE) versus the CFE antigens were not observed by the ELISA assay, and no specific humoral response was detectable in the serum of the animals before the prime immunization or in the control group.However, the serum samples of both experimental groups showed statistically the same recognition of both the parasite extracts.
The evaluation of the allergenic properties by Passive Cutaneous Anaphylaxis (PCA) assay allowed for the visualization of profound localized allergic reactions triggered by allergen-induced cross-linking of the FcRI by the binding of allergen-specific IgE located just beneath the skin.The extravasation of Evans blue dye reflected the increase in local vascular permeability, a process that depends on the release of histamine and serotonin mast cell degranulation.The PCA tests were tested by CBE in rats sensitized with anti-CBE serum to indicate the allergenic property of this parasite extract (Figure 2).No reactions were observed in the control serum area or in the CFE tested rat.In the recognition of immunogenic proteins by immunoblotting most bands were observed between 80 and 15 kDa (CPE) or 70 and 10 kDa (CBE) in the SDS-PAGE.The sharpest CPE band was near to 80 kDa.However, specific IgG recognized CPE proteins with 120 kDa or more, near to 80kDa (sharpest band), 60 kDa, 50 kDa, near 32k, 30 kDa and 25 kDa (Figure 3).However, specific IgG different recognized CBE bands from 120 kDa to 24 kDa with the sharpest bands at 85 kDa, near to 57 kDa, 35kDa and 24kDa.No reactivity was observed in the control serum.
The immunogenic capacities of the CPE and CBE after the first, second and third i.p. inoculations were shown by ELISA, with detectable high levels of specific IgG and continuously increasing IgE up to the end of the experiment.Our results corroborate previous data that indicate the use of the murine model with BALB/c mice, testing by i.p. antigenic administration, as appropriate for identifying and characterizing allergens with a protein nature (Rodero & Cuéllar, 1999;Dearman & Kimber, 2001;Vázquez-López et al., 2001;Martínez de Velasco et al., 2002;Gòmez-Morales et al., 2008;van der Ventel et al., 2011).
The cross-reactions observed by ELISA for the CPE and CBE antigens suggest that the two extracts share antigenic recognition sites.Previous studies had discarded the blastocysts and used only the plerocerci, but natural exposure may involve both portions of the metacestodes.The difference between the CPE and CBE antigens with respect to the responses induced was only statistically significant on days 14, 21 (IgE), 42, 49 (IgG) and 120 (IgG and IgE) after the first immunization.In general, CBE induced higher titers of IgG and IgE, but all immunized groups were statistically extremely different from the control group as from 14 days after the first immunization (p<0.001) for both immunoglobulins.
The ELISA and PCA results indicated the allergenic nature of CPE and CBE, since high IgE and IgG (mainly IgG1) levels are known to be related to the regulation of hypersensitivity reactions (Rodero & Cuéllar, 1999;Vázquez-López et al., 2001;Martínez de Velasco et al., 2002).
The SDS-PAGE and Western blot profile of P. crassicolle showed similar aspects when compared with other Trypanorhyncha cestodes such as G. gigas and M. horridus, which also presented IgG binding proteins with similar weight.Vázquez-López et al. (2002) observed a 24 kDa collagenase of G. gigas which as recognized by the humoral response of the experimental animals, and Gòmez-Morales et al. (2008) reported IgG binding proteins from M. horridus with 26 and 75 kDa.These proteins could be closely related to the IgG binding proteins of P. crassicolle.Since our results indicated the allergenic activity of P. crassicolle antigens in murine models, complementary clinical trials are required to elucidate their implications with respect to human health.

Figure 1 .
Figure 1.Dynamics of the specific IgG (A) and IgE (B) serum levels.Two groups, each with 7 mice, received intraperitoneally 50 µg of crude extract of Pterobothrium crassicolle plerocerci -CPE (square) or blastocysts -CBE (triangle) associated with 2 mg Al(OH) 3 , on days 0, 35 and 120 (arrow).A control group (circle) with 5 animals, received saline solution with 2 mg Al(OH) 3 on the same days using the same pathway.The values indicate the means of the sums of the optical densities (OD).+/-standard error of the mean of each group.As from day 14, the IgG and IgE levels of both experimental groups were p<0.001 (exception *p<0.01) when compared to the control.a p<0.05, b p<0.01, c p<0.001 between groups.

Figure 2 .
Figure 2. The passive cutaneous anaphylaxis assay (PCA).PCA reaction using Lou-H rat as the receptor of anti-CBE sera from BALB/c mice.Positive PCA reactions for mice sera after 127 (a) and 135 (b) days induced by the CBE.Increased blood influx (arrows); Sera without reaction (asterisks); Crude extract of Pterobothrium crassicolle blastocysts (CBE).Bar = 10 mm.